Effects of seed priming mediated by iron oxide magnetic nanoparticles (MNPs) in Capsicum annuum
Effects of seed priming mediated by iron oxide magnetic nanoparticles (MNPs) in Capsicum annuum
RNA extraction was performed on a total of 10 seeds for each experimental condition. Three independent biological replicates were included for each treatment, comparing the control group (seeds in MilliQ water for 24 h) with the treatment group exposed to 100 mg/L MNP for 24 h. Since pepper seeds were the vegetal material used for the experiment, we didn't use any growth protocol. Seeds were incubated with 100 mg/L NP and placed on a rotator at room temperature in the dark for 24 hours. Control seeds were kept in MilliQ water and maintained as treated seeds. After the treatment, seeds were rinsed in distilled water and placed in 10 cm Petri dishes on a filter soaked in 2 ml of distilled water. RNA was extracted from ten seeds using 1 mL of TRIzol extraction buffer (Thermo Fisher Scientific, Wilmington, DE, United States) according to the manufacturer's protocol. RNA concentration was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). The RNA extracted from each sample was treated with DNaseI (NEB) and purified using specific silica-based method following the manufacturer instruction (Monarch RNA Cleanup kit – NEB). The quality of the processed extracts was assessed using Bioanalyzer 2100 system (Kit RNA 6000 Nano - Agilent Technologies). Libraries were processed according to Illumina mRNA Library Prep kit instructions and a unique dual index combination was used for each sample for barcoding. The concentration and quality of the libraries were evaluated using Qubit 4 Fluorometer (dsDNA High Sensitivity Kit - Invitrogen) and Bioanalyzer 2100 system (HS DNA kit – Agilent Technologies).